Saliva is the principle protective agent for the mouth and thus is of primary importance to oral health maintenance. Perturbations of salivary secretory mechanisms can consequently lead to serious oral health problems. The objective of this project is to study the membrane and cellular processes that underlie the phenomenon of salivary fluid secretion and thus to contribute to our understanding of the fluid secretory process. Because similar secretory mechanisms are thought to be common to a number of other tissues, this information should be of rather broad applicability and interest. During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1). This plasma membrane transport protein is thought to be the major Cl entry pathway into salivary acinar cells and thus to be primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of this protein and its behavior in acinar cells will improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. Over the past year we have concentrated on three projects: (i) Characterization of possible intramolecular interactions in NKCC1. There is now good evidence that the central hydrophobic domain of NKCC1 contains the regions primarily responsible for ion transport and substrate affinity, while the intracellular termini are thought to be primarily involved in transport regulation. We hypothesize that regulatory changes in the NKCC1 termini (e.g., phosphorylation) must be transmitted to the membrane spanning domain through some sort of intra-molecular interactions and that therefore some of the intracellular regions of NKCC1 must necessarily interact with one another. To test this hypothesis we have employed the yeast two-hybrid-system using the NKCC1 termini and intracellular loops as both bait and prey. Thus far we have found evidence for the interaction of the N terminus with itself and with the C terminus as well as several possible interactions between the termini and intracellular loops. We are now in the process of confirming these interactions in intact cells using a novel method based on fluorescence resonance energy transfer. (ii) Identification and characterization of the functional regions of NKCC1. In order to identify and characterize the functional regions of NKCC1 we are carrying out cysteine scanning mutagenesis. The principle of this method is to replace amino acids at or close to possible functional residues with cysteine and then to evaluate the effects of reacting the mutated protein with sulfhydryl reagents. Since highly specific sulfhydryl reagents with a wide range of physical properties are available, it is possible to obtain considerable information about the environment of reacting sites. We have begun by making conservative mutations (alanine or serine to cysteine) in regions of NKCC1 that we expect to be accessible from the extracellular solution but close to the membrane. One of these mutants (A483C) has proven to be particularly interesting since it renders the protein sensitive to inhibition by several sulfhydryl reagents that have no effect on the wild type transporter. This residue is located in membrane spanning region 6, a highly conserved portion of the protein that our previous studies indicate is critical for function. (iii) Further studies of the membrane integration of NKCC1. In previous studies we described a series of experiments using in vitro translation in the presence of canine pancreatic microsomes to determine the location and properties of the membrane spanning segments of NKCC1. Briefly stated, this method employed expression vectors that appended a portion of the beta subunit of the H+/K+-ATPase containing multiple glycosylation sites onto the C terminal end of sequences being tested for signal anchor or stop transfer activity. Since the glycosidation of this peptide could be readily detected by a ~14 kDa shift in the apparent molecular weight of the resulting recombinant protein, the integration and orientation of the putative membrane spanning segments in the microsome could be easily determined. We have now carried this method over to an intact cell system that allows us to explore the behavior of much larger recombinant proteins (the in vitro translation system is limited to 2-3 membrane spanning segments) in a much more biologically relevant environment (intact cells vs. isolated microsomes). We are presently using this system to explore the membrane integration of both NKCC1 and the water channel AQP1.